The present invention relates to improved apparatus and methods for performing qualitative and quantitative analysis of microscopic biological specimens. In particular, the invention relates to such apparatus and methods for isolating, collecting, immobilizing, and/or analyzing microscopic biological specimens or substances which are susceptible to immunospecific or non-specific binding with magnetic-responsive particles having a binding agent for producing magnetically-labeled species within a fluid medium. As used herein, terms such as “magnetically-labeled specimen” shall refer to such biological specimens or substances of investigational interest which are susceptible to such magnetic labeling.
U.S. Pat. No. 5,985,153 describes an apparatus and method wherein an external magnetic gradient is employed to attract magnetically labeled target specimens present in a collection chamber to one of its surfaces, and where an internal magnetic gradient is employed to obtain precise alignment of those specimens on that surface. The movement of magnetically labeled biological specimens to the collection surface is obtained by applying a vertical magnetic gradient to move the magnetically labeled biological specimens to the collection surface. The collection surface is provided with a ferromagnetic capture structure, such as plurality of ferromagnetic lines supported on an optically transparent (viewing) face of a sample chamber.
Once the magnetically labeled biological specimens are pulled sufficiently close to the surface by the externally applied gradient, they come under the influence of an intense local gradient produced by the ferromagnetic collection structure and are immobilized at positions laterally adjacent thereto. The local gradient preferably exceeds adhesion forces which can hold the biological specimens to the transparent surface after they collide with the surface. Alternatively, the adhesiveness of the surface must be sufficiently weak to allow the horizontal magnetic force to move the magnetically labeled biological specimens towards the ferromagnetic structures. The smoothness and the hydrophobic or hydrophilic nature of the surface are factors that can influence the material chosen for the collection surface or the treatment of this surface to obtain a slippery surface.
U.S. Ser. No. 10/733,829 and U.S. Pat. No. 6,790,366 describe methods and apparatus for separating, immobilizing, and quantifying biological substances in a fluid sample, incorporating the principles of the externally applied gradient described above, and further incorporate a high internal gradient magnetic capture structure on the transparent collection wall. The capture structure encourages a uniform alignment of caputred biological substances for quantitative analysis with automated enumeration techniques.
In accordance with the present invention, there are described further alternative embodiments and improvements for the collection chamber whereby the internal magnetic capture sturcture is used in conjunction with small V-shaped grooves on the fluid side of the optically transparent (viewing) face of the chamber to align the target specimens for automated optical analysis. A prefereed embodiment of the present invention replaces the internal magnetic capture structure with small V-shaped grooves on the fluid side of the optically transparent (viewing) face of the chamber, and with the optimum dilution of magnetically-labeled specimens provides an alignment surface for automated optical analysis. In both embodiments, magnetically-labeled specimens and unbound magnetic particles move toward the inner surface of the chamber's viewing face, under the influence of the externally applied magnetic gradient. When they approach the surface, they come in contact with the slope of the V-shaped groove, forcing the magnetically-labeled specimens and unbound magnetic particles to move to the top of the groove. At the top of the V-shaped groove is a small chimney-shaped component with a width of approximately 2 to 3 μm which stops the magneticall-labeled specimens and allows the unbound magnetic particles to move further up into the chimney structure and outside the focal plane, used in optical analysis. This allows for alignment of the cell population in a profile that allows easier scanning with minimization of nonhomogenously illuminated cell and provides an image of the cells without the interferring ferrofluid. In the preferred embodiment, the need for internal magnetic capture structures, previously described, is not present, thus reducing the overall manufacturing cost of the viewing chamber.